Various methods are known in the art for the synthesis of nucleotides or oligonucleotides with a modified phosphate moiety. Synthetic (deoxy) oligonucleotides are usually prepared on a solid phase with the aid of phosphoramidite chemistry. Glass beads with pores of a defined size are usually used as the solid phase (abbreviated in the following as CPG= controlled pore glass). The first monomer is linked to the support by a cleavable group so that the free oligonucleotide can be cleaved off after completion of the solid phase synthesis. In addition the first monomer contains a protected hydroxyl group in which case dimethoxytrityl (DMT) is usually used as the protective group. The protective group can be removed by acid treatment. Then at the 5′ end 3′ phosphoramidite derivatives of (deoxy) ribonucleosides that are also provided with a DMT protective group are successively coupled to the reactive group that is freed in each case of the DMT protective group in a cyclic process. Alternatively 3′ dimemoxytrityl-protected 5′ phosphoramidites are used in inverse oligonucleotide synthesis. The H-phosphonate strategy is also used in particular to introduce modifications on the phosphate backbone, e.g., to prepare radiolabeled phosphorothioates. Various strategies are also already known for preparing modified or labeled oligonucleotides: trifunctional support materials are used according to the prior art to prepare oligonucleotides labeled at the 3′ end (U.S. Pat. Nos. 5,290,925, 5,401,837). Labeled phosphoramidites in which the labeling group is bound to the phosphoramidite via a C3-12 linker are usually used to prepare oligonucleotides labeled at the 5′ end (U.S. Pat. Nos. 4,997,928, 5,231,191). Furthermore modifications can be introduced into oligonucleotides on the individual bases (U.S. Pat. Nos. 5,241,060, 5,260,433, 5,668,266) or by introducing internal non-nucleoside linkers (U.S. Pat. Nos. 5,656,744, 6,130,323).
Alternatively an internucleoside phosphate can be labeled by postsynthetic labeling of phosphorothioates (Hodges, R. R., et al., Biochemistry 28 (1989) 261-7) or by post-labeling a functionalized phosphoramidite (Agrawal, S., Methods in Mol. Biology 26 (1994) Protocols for Oligonucleotide Conjugates, Chapter 3; Humana Press, Totowa, N.J.). However, these methods have not gained acceptance due to the instability of the phosphoramidites and phosphoric acid thioesters.
It is also already known from the prior art that modifications can be introduced on the inter-nucleoside phosphate residue of oligonucleotides. In the most prominent cases these are phosphothioates (Burgers, P. M., and Eckstein, F., Biochemistry 18, (1979) 592-6), methylphosphonates (Miller, P. S., et al., Biochemistry 18 (1979) 5134-43) or boranophosphates (WO 91/08213). Special monomers have to be synthesized in order to prepare methylphosphonate oligonucleotides. In contrast conventional phosphoramidites or H-phosphonates can be used to synthesize phosphorothioates and boranophosphates in which case the borano or thio modification can be introduced directly during or also after oligonucleotide synthesis by using special reagents that react with the H-phosphonate or with the phosphonic acid triester. Although all these methods lead to modified oligonucleotides, the requirements of the synthesis chemistry used for this does not allow labels that can be detected in this manner or functional groups to be directly introduced on the phosphate backbone of the oligonucleotide chain during oligonucleotide synthesis.
Baschang, G., and Kvita, V., “Angewandte Chemie” 85(1) (1973) 43-44 describe the reaction of a nucleotide phosphoric acid triester with azides such as methylsulfonyl azide to prepare tri-alkyl(aryl)imidophosphates which are, however, unstable and decompose.
Nielsen, J., and Caruthers, M. H., J. Am. Chem. Soc. 110 (1988) 6275-6276 describe the reaction of deoxynucleoside phosphites provided with a 2-cyano-1,1-dimethylethyl protective group in the presence of alkyl azide. Furthermore, the authors suggest that this principle is suitable for preparing nucleotides that are modified on the phosphate residue without elucidating which types of modifications prepared with the aid of the disclosed method could have particular advantages. In particular the authors suggest the introduction of alkyl residues.
WO 89/09221 discloses N-alkyl phosphoramidites or rather oligonucleotides substituted with N-alkyl on at least one phosphate residue which are prepared by oxidizing nucleoside phosphites (provided with a protective group) with iodine in the presence of suitable alkylamines.
WO 03/02587 discloses the preparation of modified oligonucleotides in which H-phosphates are converted by amination into phosphoramidates.
Thus all of these publications describe the preparation of molecules which contain a phosphoramidate instead of a phosphate residue. However, molecules containing phosphoramidate are susceptible to hydrolysis since the amine group is protonated in an acidic environment and is then substituted by water.
In addition WO 01/14401 proposes nucleotide building blocks or oligonucleotides in which a phosphate residue is substituted with N—ClO3, N—NO2 or N—SO2R. According to the teaching from WO 01/14401 such substances can be prepared by reacting the free hydroxyl group of a deoxy nucleoside with amidophosphonyl chloride in the presence of pyridine. However, this type of preparation is complicated, time-consuming and unsuitable for the routine synthesis of nucleotides or oligonucleotides.
The preparation of Acc azides such as acyl azides and sulfonyl azides is simple and known for a long time (Review. Brüse, S., et al., Angewandte Chemie 117 (2005) 5320-5374, 3.4 and 3.5.2). They are preferably prepared from acyl chlorides or sulfonyl chlorides using sodium azides or from hydrazides using nitrous acid.
Dye sulfonyl azides are for example also used in dyeing processes (e.g. DE 19650252). Cyanogen azide can be simply produced by reacting sodium azide with bromocyanogen in acetonitrile (McMurry, J. E. et al., J. Organic Chemistry 38(16) (1973) 2821-7). Heteroaryl azides can be prepared by nucleophilic substitution of a halogen with azide or from heteroaryl hydrazines. A prerequisite is that the electron-attracting nitrogen is in the para or ortho position relative to the azido group since only then is a resonance-stabilized phosphate mimetic formed. Ortho and para N-alkyl pyridinium azides are particularly suitable in this connection. Some acyl, sulfonyl and pyridyl azides are also commercially available.
The technical object forming the basis of the present invention was thus to provide improved labeled oligonucleotides and to provide labeling reagents which can be used within a simple process for their preparation.